PROJECT SUMMARY Methods to increase or protect beta-cell mass in vivo would have a substantial impact on T1D, where beta cells are selectively targeted for autoimmune destruction. The discovery of novel small molecules represents an attractive opportunity to protect and increase beta-cell mass in vivo, as chemical compounds provide temporal control, tunability, cell permeability, and reversibility. However, current methods to identify new compounds are relatively ad hoc and small in scale. These limitations have led to compromises in the number of compounds that can be screened. Here, we propose to leverage our expertise in chemical and cell biology to develop a high- throughput platform (?one-compound, one-islet?) for small-molecule discovery that enables the use of only a single islet to test each compound. Such an advance will allow us to screen many more compounds with each islet shipment, reducing the number of donors required, and increasing scientific rigor substantially by supporting multiple donors to be tested per compound as well. We will adapt three existing readouts for this purpose. First, we have previously developed a zinc-catalyzed prodrug system for beta cell-selective compound delivery. To measure proliferation, we will extend this synthetic system to the thymidine analog 5-ethynyl-2?-deoxyuridine (EdU). A beta cell-selective EdU will reduce the background from other proliferative cells and enable us to image intact islets after small-molecule treatment. For beta-cell survival, we will measure caspase-3 activation, which is specific to beta cells after cytokine treatment. Finally, to measure insulin secretion, we have developed a novel fluorescence-based assay that will allow us to monitor insulin secretion by imaging islets. The successful outcome of this proposal is a platform of small-molecule assays and chemical tools for understanding and promoting beta-cell regeneration and survival. A key advantage of this proposal is that the phenotypic readouts have already been fully developed, and will benefit tremendously from miniaturization to single-islet format. We are committed to making this platform available to the HIRN community, enabling investigators a) to probe their own compounds in this single-islet format, or b) to use a new readout to evaluate compounds screened in this project.